Sensitive and specific chemical labels or signal groups are widely used in chemical analysis. These labels include radioactive atoms, fluorescent reagents, luminescent molecules, metal-containing compounds, electron absorbing substances, and light absorbing compounds. In each case, one or more techniques are available to measure the particular label of interest. For example, in the case of electron-absorbing labels, measurements can be carried out by gas chromatography with electron capture detection (GC-ECD).
Not all analytical procedures involve the use of such chemical labels, but generally those applicable procedures can be divided into three broad categories. In the first category, the substance to be measured (analyte substance or analyte) is reacted with the label during the analytical procedure, leading to a labeled analyte. The signal from this labeled analyte then provides a measurement of the analyte substance. In the second category, the analyte in the sample is not labeled, but a labeled internal standard, labeled comparison substance, or labeled specifc binding partner is employed in the procedure. An example of the second category is the use of chemical tracers in radioimmunoassay or immunoradiometric assay procedures. The third analytical category is exemplified by the double isotope derivative technique. This technique involves both labeling of the analyte and the use of one or more labeled internal standards. The labeled internal standard substances may be labeled additionally in this isotope derivative procedure along with the analyte.
There are major shortcomings associated with each of the types of chemical labels currently employed in analytical procedures. For example, the use of radiolabels, particularly the more sensitive radiolabels like .sup.125 I, is limited by their short half-lives; by the physical instability and tendency for chemical lability with these labels; by safety and disposal considerations; and by the unavailability of several, closely related forms which can be measured simultaneously with comparable sensitivity and complete discrimination. Radiolabels like .sup.3 H or .sup.14 C are limited in these same respects (except for the longer half-lives of .sup.3 H and .sup.14 C), and are limited additionally by their lower sensitivity and by the susceptibility of the beta signals from these labels to quenching in the sample or liquid scintillation matrix used for counting of the label.
Many of these same limitations also apply to the use of other types of labels, particularly the problem that the magnitude of the signal from these nonradioactive labels tends to depend on the molecular environment of the label, including substances that are bound to the label covalently. Thus, it is generally important to minimize differences in the sample matrix (composition of background substances in the sample) when nonradioactive labels are being employed. This is not always controlled adequately, potentially leading to a loss in accuracy and precision of the analysis. However, it can be useful in certain analytical procedures that the signal from a label is sensitive to the molecular environment of the label, e.g., in fluorescence polarization ligand assays.
Another general limitation of currently available chemical labels is the loss in the assay sensitivity at some point when the sample of interest is progressively diluted to larger volumes prior to measurement of the signal associated with the label. This occurs because analytical procedures typically involve dilution steps arising from the addition of analytical reagents and solutions to the sample undergoing analysis, or from chromatographic separation steps, which generally, in the absence of enrichment mechanisms, cause dilution of the sample.
A particular shortcoming in the measurement of the class of labels called "electron absorbers", which are detected by their ability to absorb electrons in the vapor state, is that these labels have generally been employed only to measure molecules which are inherently volatile, or volatile after a labeling step. The most common technique for measuring molecules which contain electron-absorbing groups as labels is gas chromatography with electron capture detection (GC-ECD). In this technique, the sample to be analyzed is first injected into a gas chromatography column. The components in the sample are then separated in the volatile state by passage through the column. Finally, these components are detected based on their ability to capture gaseous electrons which comprise or influence an electrical current in an electron capture detector located at the exit of the column.
Label or signal groups frequently are combined with reactivity groups in order to allow covalent attachment of the label to the substance of interest. For example, a Bolton Hunter reagent is available commercially in which an .sup.125 I radiolabel is incorporated into a reactive molecule of p-hydroxyphenyl-propionic acid active ester. This reactive labeling reagent is used especially to radiolabel peptides and proteins with .sup.125 I.
The use of reactive, electron-absorbing labeling reagents in chemical analysis has been reviewed recently (Analytical Chemistry 52, 1002A (1980)). These reagents are used to derivatize analytes to increase the sensitivity and volatility of the analytes for analysis by GC-ECD.
Label or signal groups have not been combined, however, with both reactivity and chemical release groups. These latter groups are defined as molecular groups which are specifically released by certain chemical reaction conditions to separate the signal group from the substance to which the labeling reagent has been attached. Two common examples of specific chemical release groups are methionylamides, which are split by cyanogen bromide; and 1,2-diol (vic-glycol) groups, which are split by periodate. The applications of methionylamide cleavage comprise generation of peptide fragments for sequencing (Methods in Enzymology, 11, 238 (1967)); removal of acylmethionine protecting groups in peptide synthesis (Biochemistry 13, 5159 (1974)), and Biochemical Journal 165, 479 (1977)); and polypeptide uncoupling in protein synthesis by recombinant DNA techniques (Science, 198, 1056 (1977)).
A radiolabeled or otherwise labeled Edman reagent has been used to sequence polypeptides (see J. Biol. Chem, 250, 3629 (1975)); a process involving a release step. However, such Edman reagents do not incorporate a release group. The opportunity for release arises as a consequence of the attachment of the Edman reagent to a peptide of peptide equivalent. Splitting takes place at a site on the peptide near the attached Edman group, rather than within the attached Edman group. This applies as well to an Edman reagent which incorporates an electron absorbing group (Proc. Soc. Exp. Biol. Med., 155, 287 (1977)).
A class of reagents called "protecting groups" are widely employed in peptide synthesis. These reagents are reactive, a few of them possess groups which can be detected, and these reagents ultimately are removed from the peptide after it is synthesized. However, protecting groups differ from release tags both functionally and structurally. The purpose of protecting groups is to facilitate synthesis rather than analysis, and their removal from the peptide, after this peptide is synthesized, necessarily involves a breakage of the bond previously made to the peptide by the reactivity group. Usually chemical cleavage is performed, but an ezyme-labeled protecting group also has been used (Proceedings National Academy of Sciences 72, 2193 (1975)).
In one case a signal group (phenylazo residue) was incorporated into a protecting group for peptide synthesis, allowing one to monitor colorimetrically or visually the purification of the protecting group-peptide adduct (Helv. Chim. Acta 41, 491 (1958)), in German; summarized in English on pages 17-18 in "The Peptides", Vol. 3, E. Gross and J. Meienhofer, Academic Press, 1981. However, this monitoring is performed without release of the signal group. Thus, one of the useful chemical conditions presented for removing the protecting group acceptably causes degradation and loss of color of the signal group.
A binding assay employing an enzyme-cleavable substrate as a label involving a conjugate compound has been introduced with the conjugate comprising the four-part structure "(galactosyl)-(umbelliferone)-(linking group)-(binding component)" (see U.S. Pat. Nos. 4,226,798 and 4,279,992). Enzymatic cleavage at the (galactosyl)-(umbelliferone) bond increases the intensity of the signal from the dye indicator umbelliferone group. However, there is no release of the umbelliferone signal group from the binding component, which binding component is the substance of interest.